Topological speciation of actinide-transferrin complexes by capillary isoelectric focusing coupled with inductively coupled plasma mass spectrometry: evidence of the non-closure of the lobes.

The determination of the binding constant between transferrin and thorium and the conformational changes of the protein upon metal complexation (thorium and plutonium) have been studied by both capillary electrophoresis (CE) and capillary isoelectric focusing (cIEF) coupled with inductively coupled plasma mass spectrometry (ICP-MS). This method allows the use of both the separation power of the cIEF and the low detection limit of ICP-MS which is critical when working with highly radioactive elements. To our knowledge, this is the first time a method coupling cIEF and ICP-MS is reported in the literature. Nitrilotriacetate was used to prevent from actinide hydrolysis and as a competitive ligand with transferrin. The binding constant for the complexation of transferrin and thorium, in the absence of bicarbonate at pH 7, was found to be log K = 18.65 ± 0.19. This value, close to that of transferrin with iron, evidenced the high affinity of the protein for thorium. The results obtained by the newly developed method, cIEF-ICPMS, showed no pI change after the addition of thorium or plutonium, whereas a pI shift (linked to conformational changes) occurred for the transferrin-indium complex. This suggests that, despite the high affinity towards the actinides, the protein does not undergo a significant structure change upon complexation. The important ionic radius of the cations Th4+ (1.05 Å, CN = 8) and Pu4+ (0.96 Å, CN = 8) with respect to Fe3+ (0.645 Å, CN = 6) and to a lesser extent to In3+ (0.800 Å, CN = 6) suggests that the transferrin lobe does not close completely after complexation. However, mixed indium-actinide complexes showed structural changes even at high concentrations of apotransferrin. The conformational change is not governed by the actinide but by the other metals present.